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From Flask to Vat: The Challenges of Scaling Up Gene Therapy

Over the past two decades, gene therapy has been incubating at a very small scale in academic research labs. Like a chef fine-tuning a new recipe, scientists have been working in single batches that would only be sufficient to treat one or two patients at a time. 

But now, as these treatments are beginning to show great promise for rare diseases such as Duchenne muscular dystrophy (DMD) and hemophilia, researchers are facing the next challenge: scaling up to produce gene therapy for clinical trial patients and then eventually at the commercial level, to reach thousands of real-world patients.  

“Now that this technology has demonstrated it can help patients, we need to make more and more,” says Patrick Bastek, Sr. Director, Gene Therapy Process Development at Pfizer based in Research Triangle Park, North Carolina. “A big part of it is using the technology that has already been developed and adapting it for increased scale." 

Two-liter (left), 200-L (center) and 2,000-L (right) bioreactors used in manufacturing gene therapy for Duchenne Muscular Dystrophy. A primary focus of gene therapy process development scientists like Pfizer’s Mensah and Bastek is to determine how to develop these therapies at progressively larger scales so that there’s a sufficient amount for both clinical trials and for commercial use. 

A process to enable the product  

Similar to the process of scaling up production for vaccines and biological medicines, creating gene therapies is a complex undertaking that involves engineering live cells to manufacture therapeutic proteins. “But with gene therapy, there are some unique challenges because it’s so new and there are so many more parts that go into it,” says Bastek. The finished product is the outer shell, or capsid, of an adeno-associated virus (AAV), engineered to remove its genetic material. Because the AAV capsid contains none of the virus’ original genes, the capsid can instead act as a vehicle to deliver copies of a therapeutic gene to a patient’s cells.   

As Pfizer has recently initiated clinical trials for a DMD gene therapy, scientists are working to develop novel manufacturing processes to produce AAV vectors to treat DMD. “We’re essentially developing processes, so we can move the product seamlessly along the development path from clinical to commercial manufacturing,” says Paul Mensah, the head of Pfizer’s Bioprocess R&D, based in St. Louis, Missouri. The goal, he says, is that the process developed can also be applied to other, future clinical programs.

Finding a winner  

There are a variety of established strategies to manufacture AAV in very small quantities, but now scientists are faced with the challenge of adapting these approaches to make clinical grade product on a larger scale. Since different methods might be appropriate for different treatments, Pfizer is currently working to develop multiple platforms to develop AAV. “Out technological development effort is to study a few approaches in detail to see if we can find a winner," says Mensah.  

But Pfizer scientists are focusing in house on one manufacturing strategy in particular: triple transfection. This process involves introducing three different types of plasmid (a form of DNA) to instruct the cells to make parts of AAV. These plasmids are required for programming for its critical parts of AAV: the proteins that make up the shell and enable it to replicate, helper proteins that enable the vector to assemble correctly, and the vector genome that codes for the therapeutic protein (dystrophin, in the case of DMD)—hence the name triple transfection.  

“There are a lot of complex moving parts to ensure that it’s put together in such a way that the gene of interest ends up inserted in the AAV vector,” says Bastek. Once the cells grow to an appropriate density, they can manufacture the AAV vectors in three days. 

Moving to a larger scale 

To move the AAV manufacturing process from small batch to a larger scale, scientists also need to find new ways to efficiently culture cells in large containers. Typically, the cells, which in this case are human embryotic kidney (HEK) cells, grow on a surface in a plastic flask or bottle. However, to scale it up to generate sufficient material, it is best to have the ceslls in suspension—floating freely in the vessel. Bastek and his team are optimizing ways to grow the cells to very high densities in suspension cultures while maintaining the specific productivity of the cells. The result is an overall increase in culture productivity. “The approach offers significant advantage over growing cells in multiples of plastic containers,” he says.

A consistent product 

In addition to the challenges scientists face in having a large enough volume of productive HEK cells for both clinical and commercial use, they need to ensure the finished AAV capsids all contain the therapeutic gene—in other words, that they aren’t producing empty shells. “Not all of the product has the gene of interest in there, and part of our challenge is ensuring that what we’re giving the patient has a high percentage of full capsids,” says Bastek.  

At the lab scale, scientists can easily spin test tubes at a very high speed, a process called ultracentrifugation, to separate the full capsids from the empty ones. This purification step is a critical element of the overall process development that Bastek’s team focuses on—but it’s not practical for scaling up. So Pfizer scientists are currently working to develop alternative technologies to perform this operation consistently at larger scales. Key to their efforts are ways to assess the effectiveness of the step and quality of the product. “Our colleagues focused on analytical R&D have come up with some newer techniques, such as using electron microscopy and mass spectrometry to see the difference between empty and full capsids,” says Bastek. Scientists also need to develop complex assays to test the safety, purity and potency of the manufactured AAV.  

As gene therapy breakthroughs advance, scientists are now confronting the challenges of efficiently and effectively bringing these life-transforming therapies to the patients who need them.